Total cell DNA
Preparation of total cell DNA
The fundamentals of DNA preparation are most easily understood by first considering the simplest type of DNA purification procedure, that wbere the entire DNA complement of a bacterial cell is required. The modifications needed for plasmid and phage DNA preparation can then be described later.
The procedure for total DNA preparation from a culture of bacterial cells can be divided into four stages:
(1) A culture of bacteria is grown and then harvested.
(2) The cells are broken open to release their contents.
(3) This cell extract is treated to remove all components except the DNA.
(4) The resulting DNA solution is concentrated.
1) Growing and harvesting a bacterial culture
Most bacteria can be grown without too much difficulty in a liquid medium (broth culture).
The culture medium must provide a balanced mixture of the essential nutrients at concentrations that will allow the bacteria to grow and divide efficiently.
In LB medium at 37°C, aerated by shaking at 150-250rpm on a rotary platform, E. coli cells divide once every 20min or so until the culture reaches a maximum density of about 2-3 x 109 cells/ml. The growth of the culture can be monitored by reading the optical density (OD) at 600nm, at which wavelength one OD unit corresponds to about 0.8 x 109 cells/mL In order to prepare a cell extract, the bacteria must be obtained in as small a volume as possible. Harvesting is therefore performed by spinning the culture in a centrifuge. Fairly low centrifugation speeds will pellet the bacteria at the bottom of the centrifuge tube, allowing the culture medium to be poured off. Bacteria from a 1000ml culture at maximum cell density can then be resuspended into a volume of 10ml or less.
2) Preparation of a cell extract
The bacterial cell is enclosed in a cytoplasmic membrane and surrounded by a rigid cell walL With some species, including E. coli, the cell wall may itselfbe enveloped by a second, outer membrane.
All of these barriers have to be disrupted to release the cell components.
Techniques for breaking open bacterial cells can be divided into physical methods, in which the cells are disrupted by mechanical forces, and chemical
methods, where cell lysis is brought about by exposure to chemical agents that affect the integrity of the cell barriers.
Chemical methods are most commonly used with bacterial cells when the object is DNA preparation.
Chemical lysis generally involves one agent attacking the cell wall and another disrupting the cell membrane.
The chemicals that arellsed depend on the species of bacterium involved, but with E. coli and related organisms, weakening of the cell wall is usually brought about by lysozyme, ethylenediamine tetraacetate (EDTA), or a combination of both.
Lysozyme is an enzyme that is present in egg white and in secretions such as tears and saliva, and which digests the polymeric compounds that give the cell wall its rigidity.
On the other hand, EDTA removes magnesium ions that are essential for preserving the overall structure of the cell envelope, and also inhibits cellular enzymes that could degrade DNA. Under some conditions, weakening the cell wall with lysozyme or EDTA is sufficient to cause bacterial cells to burst, but usually a detergent such as sodium dodecyl sulphate (SDS) is also added.
Detergents aid the process of lysis by removing lipid molecules and thereby cause disruption of the cell membranes.
Having lysed the cells, the final step in preparation of a cell extract is removal of insoluble cell debris.
Components such as partially digested cell wall fractions can be pelleted by centrifugation, leaving the cell extract as a reasonably clear supernatant.
3) Purification of DNA from a cell extract
In addition to DNA, a bacterial cell extract contains significant quantities of protein and RNA. A variety of methods can be used to purify the DNA from this mixture.
One approach is to treat the mixture with reagents which degrade the contaminants, leaving a pure solution of DNA.
Other methods use ion-exchange chromatography to separate the mixture into its various components, so the DNA is removed from the proteins and RNA in
the extract.
(i) Removing contaminants by organic extraction and enzyme digestion
The standard way to deproteinize a cell extract is to add phenol or a 1: 1 mixture of phenol and chloroform. These organic solvents precipitate proteins
but leave the nucleic acids (DNA and RNA) in aqueous solution. The result is that if the cell extract is mixed gently with the solvent, and the layers then
separated by centrifugation, precipitated protein molecules are left as a white coagulated mass at the interface between the aqueous and organic layers. The aqueous solution of nucleic acids can then be removed with a pipette .
With some cell extracts the protein content is so great that a single phenol extraction is not sufficient to purify completely the nucleic acids. This problem
could be solved by carrying out several phenol extractions one after the other, but this is undesirable. as each mixing and centrifugation step results in a
certain amount of breakage of the DNA molecules. The answer is to treat the cell extract with a protease such as pronase or proteinase K before phenol
extraction.
These enzymes break polypeptides down into smaller units, which are more easily removed by phenol.
Some RNA molecules, especially messenger RNA (mRNA), are removed by phenol treatment, but most remain with the DNA in the aqueous layer. The only effective way to remove the RNA is with the enzyme ribonuclease, which rapidly degrades these molecules into ribonucleotide subunits.
(ii) Using ion-exchange chromatography to purify DNA from a cell extract
Biochemists have devised various methods for using differences in electrical charge to separate mixtures of chemicals into their individual components.
One of these methods is ion-exchange chromatography, which separates molecules according to how tightly they bind to electrically charged particles
present in a chromatographic matrix or resin.
DNA and RNA are both negatively charged, as are some proteins, and so bind to a positively charged resin. The electrical attachment is disrupted by salt, removal of the more tightly bound molecules requiring higher concentrations of salt. By gradually increasing the salt concentration, different types of molecule can be detached from the resin one after another.
The simplest way to carry out ion-exchange chromatography is to place the resin in a glass or plastic column and then add the cell extract to the top. The extract passes through the column. and because this extract contains very little salt all the negatively charged molecules bind to the resin and are retained in the column. If a salt solution of gradually increasing concentration is now passed through the column, then the different types of molecule will elute (i.e. become unbound) in the sequence protein, RNA and finally DNA.
4) Concentration of DNA samples
Organic extraction often results in a very thick solution of DNA that does not need to be concentrated any further.
Other purification methods give moredilute solutions and it is therefore important to consider methods for increasing the DNA concentration.
The most frequently used method of concentration is ethanol precipitation.
In the presence of salt (strictly speaking, monovalent cations such as
sodium ions), and at a temperature of -200C or less, absolute ethanol efficiently precipitates polymeric nucleic acids.
With a thick solution of DNA the ethanol can be layered on top of the sample, causing molecules to precipitate at the interface. A spectacular trick is to push a glass rod through the ethanol into the DNA solution. When the rod is removed, DNA molecules adhere and can be pulled out of the solution in the form of a long fibre.
Alternatively, if ethanol is mixed with a dilute DNA solution, the precipitate can be collected by centrifugation, and then redissolved in an appropriate volume of water.
Ethanol precipitation has the added advantage of leaving short-chain and monomeric nucleic acid components in solution. Ribonucleotides produced by ribonuclease t~eatment are therefore lost at this stage.
5) Measurement of DNA concentration
It is crucial to know exactly how much DNA is pfe~'ent in a solution when carrying out a gene cloning experiment.
Fortunately DNA concentrations can be accurately measured by ultraviolet (UV) absorbance spectrophotometry. The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of DNA in the sample. Usually absorbance is measured at 260nm, at which wavelength an absorbance (A26o) of 1.0 corresponds to 50 micrograms of double-stranded DNA per mL.
Ultraviolet absorbance can also be used to check the purity of a DNA preparation. With a pure sample of DNA the ratio of the absorbances at 260nm and 280nm (A26o/Az8o) is 1.8. Ratios of less than 1.8 indicate that the preparation is contaminated, either with protein or with phenol.
Other methods for the preparation of total cell DNA
Bacteria are not the only organisms from which DNA may be required.
Total cell DNA from, for example, plants or animals will be needed if the aim of thegenetic engineering project is to clone genes from these organisms. Although the basic steps in DNA purification are the same whatever the organism, some modifications may have to be introduced to take account of the special features of the cells being used.
Obviously growth of cells in liquid medium is appropriate only for bacteria, other microorganisms, and plant and animal cell cultures.
The major modifications, however, are likely to be needed at the cell breakage. The chemicals used for disrupting bacterial cells do not usually work with other organisms: lysozyme, for example, has no effect on plan t cells.
Specific degradative enzymes are available for most cell wall types, but often physical techniques, such as grinding frozen material with a mortar and pestle, are more efficient.
On the other hand, most animal cells have no cell wall at all, and can be lysed simply by treating with detergent.
Another important consideration is the biochemical content of the cells from which DNA is being extracted. With most bacteria the main biochemicals present in a cell extract are protein, DNA and RNA, so phenol extraction and/or protease treatment, followed by removal of RNA with ribonuclease,
leaves a pure DNA sample.
These treatments may not, however, be sufficient to give pure DNA if the cells also contain significant quantities of other biochemicals.
Plant tissues are particularly difficult in this respect as they often contain large amounts of carbohydrates that are not removed by phenol extraction. Instead a different approach must be used.
One method makes use of a detergent called cetyltrimethylammonium bromide (CTAB), which forms an insoluble complex with nucleic acids. When
CTAB is added to a plant cell extract the nucleic acid-CTAB complex precipitates, leaving carbohydrate, protein and other contaminants in the supernatant. The precipitate is then collected by centrifugation and resuspended in 1 M sodium chloride, which causes the complex to break down.
The nucleic acids can now be concentrated by ethanol precipitation and the RNA removed by ribonuclease treatment.
The need to adapt organic extraction methods to take account of the biochemical contents of different types of starting material has stimulated the search for DNA purification methods that can be used with any species. This is one of the reasons why ion-exchange chromatography has
become so popular.
A similar method involves a compound called guaniiainium thiocyanate, which has two properties that make it useful for DNA purification. First, it denatures and dissolves all biochemicals other than nucleic acids and can therefore be used to release DNA from virtually any type of cell or tissue. Second, in the presence of guanidinium thiocyanate, DNA binds tightly to silica particles. This provides an easy way of recovering the DNA from the denatured mix of biochemicals.
One possibility is to add the silica directly to the cell extract but, as with the ion-exchange methods, it is more convenient to use a chromatography column. The silica is placed in the column and the cell extract added. DNA binds to the silica and is retained in the column, whereas the denatured biochemicals pass straight through. After washing away the last contaminants with guanidinium thiocyanate solution, the DNA is recovered by adding water, which destabilizes the interactions between the DNA molecules and the silica.
Sources:
Content:
Gene Cloning and DNA Analysis, an intoduction(fifth edition), T. A. Brown
Images:
https://www.labome.com/method/DNA-extraction-and-purification.html
https://openwetware.org/wiki/RNA_extraction_using_trizol/tri